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Phylogenomic and MALDI-TOF MS Analysis of Streptococcus
sinensis HKU4T Reveals a Distinct Phylogenetic Clade in the
Genus Streptococcus
Jade L.L. Teng1,2,3,4,y, Yi Huang1,y, Herman Tse1,2,3,4, Jonathan H.K. Chen1, Ying Tang1,
Susanna K.P. Lau1,2,3,4,*, and Patrick C.Y. Woo1,2,3,4,*
1
Department of Microbiology, The University of Hong Kong, Hong Kong, China
2
Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong, China
3
State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China
4
Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong, China
*Corresponding author: E-mail: [email protected]; [email protected].
yThese authors contributed equally to this work.
Accepted: October 13, 2014
Data deposition: This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession JPEN00000000. The
version described in this paper is version JPEN01000000.
Abstract
Streptococcus sinensis is a recently discovered human pathogen isolated from blood cultures of patients with infective endocarditis. Its
phylogenetic position, as well as those of its closely related species, remains inconclusive when single genes were used for
phylogenetic analysis. For example, S. sinensis branched out from members of the anginosus, mitis, and sanguinis groups in the
16S ribosomal RNA gene phylogenetic tree, but it was clustered with members of the anginosus and sanguinis groups when groEL
gene sequences used for analysis. In this study, we sequenced the draft genome of S. sinensis and used a polyphasic approach,
including concatenated genes, whole genomes, and matrix-assisted laser desorption ionization-time of flight mass spectrometry to
analyze the phylogeny of S. sinensis. The size of the S. sinensis draft genome is 2.06 Mb, with GC content of 42.2%. Phylogenetic
analysis using 50 concatenated genes or whole genomes revealed that S. sinensis formed a distinct cluster with Streptococcus
oligofermentans and Streptococcus cristatus, and these three streptococci were clustered with the “sanguinis group.” As for
phylogenetic analysis using hierarchical cluster analysis of the mass spectra of streptococci, S. sinensis also formed a distinct cluster
with S. oligofermentans and S. cristatus, but these three streptococci were clustered with the “mitis group.” On the basis of the
findings, we propose a novel group, named “sinensis group,” to include S. sinensis, S. oligofermentans, and S. cristatus, in
the Streptococcus genus. Our study also illustrates the power of phylogenomic analyses for resolving ambiguities in bacterial
taxonomy.
Key words: Streptococcus sinensis, genome, phylogenomic, “sinensis group”, Illumina.
Background
The genus Streptococcus currently comprises more than 90
species with some of them being important human pathogens
causing significant morbidity and mortality globally.
Traditional phenotypic classification of Streptococcus relies
on their Lancefield group antigens and hemolytic properties,
which divides the genus into two major groups, pyogenic
and viridans groups (Sherman 1937). As a result of the
widespread use of polymerase chain reaction and DNA
sequencing in the last two decades, genotypic methods
such as amplification and sequencing of universal gene targets
represent an advanced method for bacterial classification and
identification. Among the various studied gene targets, 16S
ribosomal RNA (rRNA) gene has been the most widely
used. Classification of Streptococcus based on the 16S rRNA
gene sequences divided the genus into six major groups,
namely anginosus, mitis, salivarius, mutans, bovis, and
pyogenic
groups
(Kawamura
et
al.
1995).
ß The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits
non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected]
2930 Genome Biol. Evol. 6(10):2930–2943. doi:10.1093/gbe/evu232 Advance Access publication October 20, 2014
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Streptococcus sinensis HKU4T Genome Sequence
Subsequently, Facklam (2002) suggested the addition of sanguinis group to the existing groups based on the phenotypic
properties, resulting in seven major groups. However, some
studies still showed that 16S rRNA gene or other housekeeping genes failed to provide sufficient resolution and to delineate Streptococcus species into the same taxonomic
groupings (Tapp et al. 2003; Glazunova et al. 2010; Park
et al. 2010; Zbinden et al. 2011). Apart from these phenotypic
and genotypic identification methods, a recently emerged
technique, matrix-assisted laser desorption ionization timeof-flight mass spectrometry (MALDI-TOF MS), has been used
for identification and cluster analysis of many bacterial species.
The technique examines the profile of proteins detected directly from intact bacteria and involves pattern analysis of the
mass spectra obtained within a mass range of 2,000–20,000
Da from whole cell protein fingerprinting using mathematical
tools (Hsieh et al. 2008). Several studies have shown
that MALDI-TOF MS is useful for identification and cluster
analysis of different groups of medically important
Streptococcus species (Dubois et al. 2013; Karpanoja et al.
2014).
In 2002, we reported the discovery of a novel species,
Streptococcus sinensis, isolated from multiple blood cultures
of a 42-year-old Chinese woman with infective endocarditis
complicating her chronic rheumatic heart disease in Hong
Kong (Woo et al. 2002). Two years later, we reported two
other cases of infective endocarditis caused by S. sinensis
with Lancefield F (Woo et al. 2004). Subsequently, additional
cases reported from France and Italy suggested that the
bacterium is an emerging pathogen of global importance
(Uckay et al. 2007; Faibis et al. 2008). The oral cavity is
the natural reservoir of S. sinensis (Woo et al. 2008).
Phenotypically, the bacterium possessed characteristics from
members of anginosus, sanguinis, and mitis groups, as different commercial kits gave different identities to different strains
of S. sinensis (Woo et al. 2006). Genotypically, phylogenetic
analysis using 16S rRNA gene sequences showed that
S. sinensis was equally close to both the anginosus and mitis
groups but was more closely related to sanguinis group
when using GroEL sequences. Because of these inconclusive
results, the phylogenetic position of S. sinensis remains
uncertain.
In this study, we attempted to determine the exact
phylogenetic position of S. sinensis using phylogenomic
approach. We sequenced the first genome of S. sinensis
type strain HKU4T and performed comparative genomic
studies utilizing publicly available Streptococcus genomes.
Phylogenomic analysis using the whole-genome sequences
revealed a new phylogenetic clade, including S. sinensis,
Streptococcus oligofermentans, and Streptococcus cristatus,
which we propose it as “sinensis group.” Intergenomic
distance analysis, phylogenetic analysis using concatenated
sequences, and cluster analysis of MALDI-TOF MS were also
performed.
Genome Sequencing of S. sinensis
Type Strain HKU4T
The de novo assembly using 26.81 million paired-end reads
generated 116 contigs with lengths ranging from 212 to
125,948 bases, giving a total genome size of 2.06 Mb with
the average GC content of 42.2%. All contigs generated were
submitted to the RAST version 4.0 (Rapid Annotation
using Subsystem Technology) annotation server, resulting in
1,992 protein-coding sequences (CDSs), 4 rRNA operons,
and 47 transfer RNA-encoding genes (supplementary table
S1, Supplementary Material online). To facilitate the subsequent genomic analysis, 86 genome sequences of other
Streptococcus species and that of Enterococcus faecalis
were also submitted to RAST for annotation. Among the
86 genome sequences, 25 complete genome sequences
and 5 draft genome sequences (Streptococcus australis,
Streptococcus criceti, S. cristatus, Streptococcus dentisani,
and Streptococcus tigurinus because only one partial
genome sequence was available for each species) were used
for subsystem classification (table 1). Each CDS in annotated
genomes was grouped into different RAST subsystems based
on the predicted functional role. Among the 1,992 CDSs in
the S. sinensis HKU4T genome, 854 CDSs can be categorized
into RAST subsystems (fig. 1A), in which 133 CDSs were
classified into more than one category. Overall, majority of
CDSs were classified into subsystems of carbohydrates (148
CDSs, 7.4%), protein metabolism (118 CDSs, 5.9%), DNA
metabolism (111 CDSs, 5.6%), and amino acid and derivatives
(110 CDSs, 5.5%) (fig. 1A). The remaining 1,138 (57.1%)
CDSs could not be classified into any subsystems, in which
488 (24.5%) CDSs were only annotated as hypothetical proteins. Consistent to the results of previous studies (Olson et al.
2013), when we compared the distribution of CDSs in each
subsystem of the S. sinensis genome with those of other
Streptococcus genomes, all of them have a similar percentage
of their genome dedicated to each subsystem (fig. 1B).
Phylogenomic Analyses of S. sinensis
and Other Streptococcus Species
Reveals a Distinct Clade Comprising
S. sinensis, S. oligofermentans, and
S. cristatus
Phylogenetic analyses using sequences of single gene loci, 16S
rRNA and groEL, extracted from 87 Streptococcus genomes
showed that S. sinensis closely clustered with S. oligofermentans and S. cristatus, respectively (fig. 2A–C). However, the
topology of these trees was not concordant, in which
S. sinensis branched out from members of anginosus, mitis,
and sanguinis groups when using 16S rRNA gene sequences
(fig. 2A) but clustered with members of anginosus and
sanguinis groups when using groEL sequences for analyses
(fig. 2B and C). This inconclusive result suggested that
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2931
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Table 1
Genome Sequence Details and the Corresponding DDH Value with Streptococcus sinensis HKU4T
Species
“Sinensis” group
Streptococcus sinensis
Streptococcus oligofermentans
Streptococcus cristatus
Sanguinis group
Streptococcus sanguinis
Streptococcus sanguinis
Streptococcus gordonii
Anginosus group
Streptococcus intermedius
Streptococcus intermedius
Streptococcus intermedius
Streptococcus intermedius
Streptococcus constellatus subsp. pharyngis
Streptococcus constellatus subsp. pharyngis
Streptococcus constellatus subsp. pharyngis
Streptococcus constellatus subsp. pharyngis
Streptococcus anginosus
Streptococcus anginosus
Streptococcus anginosus
Streptococcus anginosus subsp. whileyi
Mitis group
Streptococcus oralis
Streptococcus oralis
Streptococcus mitis
Streptococcus pneumoniae
Streptococcus pneumoniae
Streptococcus pneumoniae
Streptococcus pneumoniae
Streptococcus pneumoniae
Streptococcus pneumoniae
Streptococcus pneumoniae
Streptococcus pneumoniae
Streptococcus pneumoniae
Streptococcus pneumoniae
Streptococcus pneumoniae
Streptococcus parasanguinis
Streptococcus parasanguinis
Streptococcus pseudopneumoniae
Streptococcus infantis
Streptococcus infantis
Streptococcus peroris
Streptococcus tigurinus
Streptococcus dentisani
Streptococcus australis
Pyogenic group
Streptococcus agalactiae
Streptococcus agalactiae
Streptococcus agalactiae
Streptococcus agalactiae
Streptococcus dysgalactiae subsp. equisimilis
Streptococcus pyogenes
Streptococcus pyogenes
Streptococcus pyogenes
Strain
Status
GenBank Accession No.
DDH (Formula 1)
HKU4T
AS 1.3089
ATCC 51100
Draft
Finished
Draft
JPEN00000000a,b
NC_021175.1a,b
AEVC01000001–AEVC01000031a,b
—
45.6
44.3
SK36
SK678
CH1
Finished
Draft
Finished
NC_009009a,b
AEXA01000001–AEXA01000010
NC_009785a,b
28.9
—
23.2
C270
JTH08
B196
F0413
C232
C818
C1050
SK1060
C1051
C238
SK1138
CCUG 39159
Finished
Finished
Finished
Draft
Finished
Finished
Finished
Draft
Finished
Finished
Draft
Draft
NC_022237.1b
NC_018073.1b
NC_022246.1a,b
AFXO01000001–AFXO01000013
NC_022236.1a,b
NC_022245.1b
NC_022238.1b
BASX01000001–BASX01000066
NC_022244.1a,b
NC_022239.1b
ALJO01000001–ALJO01000013
AICP01000001–AICP01000083
17.1
16.7
16.7
—
16.8
16.8
16.7
—
16.2
15.6
—
—
Uo5
SK610
B6
ATCC 700669
D39
JJA
Taiwan19F-14
TIGR4
G54
P1031
R6
70585
CGSP14
Hungary19A-6
FW213
ATCC 15912
IS7493
ATCC 700779
SK970
ATCC 700780
1366
7746
ATCC 700641
Finished
Draft
Finished
Finished
Finished
Finished
Finished
Finished
Finished
Finished
Finished
Finished
Finished
Finished
Finished
Finished
Finished
Draft
Draft
Draft
Draft
Draft
Draft
NC_015291.1a,b
AJKQ01000001–AJKQ01000031
NC_013853a,b
NC_011900a,b
NC_008533b
NC_012466b
NC_012469b
NC_003028b
NC_011072b
NC_012467b
NC_003098b
NC_012468b
NC_010582b
NC_010380b
NC_017905.1b
NC_015678.1a,b
NC_015875.1a,b
AEVD01000001–AEVD01000031
AFUT01000001–AFUT01000009
AEVF01000001–AEVF01000017
AORX01000001–AORX01000014a
CAUJ01000001–CAUJ01000008a
AEQR01000001–AEQR01000027a
16.1
—
15.4
15.4
15.4
15.4
15.4
15.4
15.3
15.3
15.3
15.2
15.2
15.0
15.2
15.1
14.8
—
—
—
—
—
—
NEM316
09mas018883
2603 V/R
A909
GGS_124
M1 GAS
Manfredo
MGAS10270
Finished
Finished
Finished
Finished
Finished
Finished
Finished
Finished
NC_004368b
NC_021485.1a,b
NC_004116b
NC_007432b
NC_012891a,b
NC_002737a,b
NC_009332b
NC_008022b
13.5
13.2
13.2
13.2
13.3
13.3
13.3
13.3
(continued)
2932 Genome Biol. Evol. 6(10):2930–2943. doi:10.1093/gbe/evu232 Advance Access publication October 20, 2014
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Streptococcus sinensis HKU4T Genome Sequence
Table 1 Continued
Species
Streptococcus pyogenes
Streptococcus pyogenes
Streptococcus pyogenes
Streptococcus pyogenes
Streptococcus pyogenes
Streptococcus pyogenes
Streptococcus pyogenes
Streptococcus pyogenes
Streptococcus pyogenes
Streptococcus pyogenes
Streptococcus uberis
Streptococcus parauberis
Streptococcus iniae
Streptococcus equi subsp. zooepidemicus
Streptococcus equi subsp. equi
Streptococcus equi subsp. zooepidemicus
Streptococcus criceti
Bovis group
Streptococcus gallolyticus
Streptococcus lutetiensis
Streptococcus pasteurianus
Streptococcus equinus
Streptococcus infantarius
Mutans group
Streptococcus mutans
Streptococcus mutans
Streptococcus mutans
Streptococcus mutans
Salivarius group
Streptococcus thermophilus
Streptococcus thermophilus
Streptococcus thermophilus
Streptococcus thermophilus
Streptococcus salivarius
Streptococcus salivarius
Streptococcus salivarius
No group
Streptococcus suis
Streptococcus suis
Streptococcus suis
Streptococcus suis
Streptococcus suis
Outgroup
Enterococcus faecalis
Strain
Status
GenBank Accession No.
DDH (Formula 1)
MGAS10394
MGAS10750
MGAS2096
MGAS315
MGAS5005
MGAS6180
MGAS8232
MGAS9429
NZ131
SSI-1
0140J
KCTC 11537
SF1
MGCS10565
4047
H70
HS-6
Finished
Finished
Finished
Finished
Finished
Finished
Finished
Finished
Finished
Finished
Finished
Finished
Finished
Finished
Finished
Finished
Draft
NC_006086b
NC_008024b
NC_008023b
NC_004070b
NC_007297b
NC_007296b
NC_003485b
NC_008021b
NC_011375b
NC_004606b
NC_012004a,b
NC_015558.1a,b
NC_021314.1a,b
NC_011134a,b
NC_012471b
NC_012470b
AEUV02000001–AEUV02000002a
13.3
13.3
13.3
13.3
13.3
13.3
13.3
13.3
13.3
13.3
13.2
13.2
13.1
13.1
13.0
13.0
—
UCN34
33
ATCC 43144
ATCC 9812
ATCC BAA-102
Finished
Finished
Finished
Draft
Draft
NC_013798a,b
NC_021900.1a,b
NC_015600.1a,b
AEVB01000001–AEVB01000057
ABJK02000001–ABJK02000022
13.3
13.3
13.3
—
—
NN2025
GS-5
LJ23
UA159
Finished
Finished
Finished
Finished
NC_013928a,b
NC_018089b
NC_017768.1b
NC_004350b
13.3
13.2
13.2
13.2
CNRZ1066
LMD-9
LMG 18311
JIM 8232
JIM8777
CCHSS3
SK126
Finished
Finished
Finished
Finished
Finished
Finished
Draft
NC_006449b
NC_008532b
NC_006448b
NC_017581.1a,b
NC_017595.1b
FR873481a,b
ACLO01000001–ACLO01000101
13.8
13.8
13.8
13.7
13.7
13.6
—
BM407
P1/7
98HAH33
05ZYH33
SC84
Finished
Finished
Finished
Finished
Finished
NC_012926b
NC_012925b
NC_009443b
NC_009442a,b
NC_012924b
13.7
13.7
13.6
13.6
13.6
V583
Finished
NC_004668.1b
12.6
a
Genome sequence submitted for subsystem classification using RAST 4.0.
Genome sequence used to construct the whole genome tree.
b
phylogenetic analysis using single gene target has not added
much to our understanding on the phylogeny of S. sinensis, as
they only represent a very small portion of the bacterial
genome.
In view of this problem, we attempted to use a
phylogenomic approach, which based on the whole bacterial
genome, to resolve the taxonomic ambiguity of S. sinensis. In
bacterial taxonomy, whole genomic DNA–DNA hybridization
(DDH) has been used to determine the genetic distance between two strains and 70% DDH was proposed as a criterion
for delineating species (Wayne 1988). However, this method is
not widely used because it is tedious and results depend on
experimental conditions. Therefore, it is often difficult to
compare results between different laboratories objectively.
Recent advancement of genome sequencing calls for bioinformatics methods to replace the wet-lab DDH by in silico
Genome Biol. Evol. 6(10):2930–2943. doi:10.1093/gbe/evu232 Advance Access publication October 20, 2014
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FIG. 1.—Distributions of predicted coding sequence function in the annotated genomes according to RAST subsystems. In (A), the number of CDSs of S.
sinensis HKU4T in different subsystems is indicated in bracket. In (B), a total of 31 genomes of Streptococcus species, including S. sinensis and representatives
from all major groups, were analyzed. Each column indicates the number of CDSs of each Streptococcus species in different subsystems showing in different
color.
2934 Genome Biol. Evol. 6(10):2930–2943. doi:10.1093/gbe/evu232 Advance Access publication October 20, 2014
Streptococcus sinensis HKU4T Genome Sequence
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FIG. 2.—Phylogenetic relationship among Streptococcus strains. Three phylogenetic trees were constructed, each using a different genetic locus for
analysis. (A) 16S rRNA. (B) groEL. (C) GroEL. The trees were constructed by maximum-likelihood method using RAxML (version 7.3) and E. faecalis V583 as
the root. A total of 1,583 nucleotide positions of the 16S rRNA gene, 1,709 nucleotide positions and 565 deduced amino acid positions of groEL from 88
genomes were included for analyses. Bootstrap values were calculated from 1,000 replicates. The scale bar corresponds to the mean number of nucleotide/
amino acid substitutions per site on the respective branch. Names and accession numbers are given as cited in GenBank in table 1.
Genome Biol. Evol. 6(10):2930–2943. doi:10.1093/gbe/evu232 Advance Access publication October 20, 2014
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FIG. 2.—Continued.
genome-to-genome comparison. Among various sophisticated methods studied, a digital DDH method, GGDC 2.0,
was shown to yield very good correlation with wet-lab DDH
(Auch et al. 2010). In this study, with the availability of
Streptococcus genome sequences, we were able to use
GGDC 2.0 for intergenomic distance estimation, which allowed genome sequence comparison between two
Streptococcus strains. The results showed that S. sinensis
HKU4T shared 45.6% and 44.3% nucleotide identities to
the genome sequence of S. oligofermentans AS 1.3089
(GenBank accession number NC_021175) and S. cristatus
ATCC 51100 (GenBank accession number AEVC01000001–
AEVC01000031) but only 23.2–28.9%, 15.6–17.1%, and
14.8–16.1% nucleotide identities to those from members of
sanguinis (3 genomes), anginosus (12 genomes), and mitis (23
genomes) groups, respectively (table 1). The present DDH
value between S. sinensis and S. oligofermentans was quite
different from the results obtained in the previous study, in
which the wet-lab DDH value between S. sinensis and S. oligofermentans was only 15%. We speculate that the
2936 Genome Biol. Evol. 6(10):2930–2943. doi:10.1093/gbe/evu232 Advance Access publication October 20, 2014
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Streptococcus sinensis HKU4T Genome Sequence
FIG. 2.—Continued.
discrepancy may due to the experimental error as DDH
method is well-known not easily be made reproducible.
Nevertheless, the present results using the entire genome sequences showed that S. sinensis was more closely related to S.
oligofermentans and S. cristatus than members of sanguinis,
anginosus, and mitis groups.
To further verify the phylogenetic position of S. sinensis
among the genus Streptococcus, phylogenomic analysis
utilizing the draft genome sequences of S. sinensis HKU4T
and S. cristatus ATCC51100 as well as the available complete
genome sequences of 69 Streptococcus species was performed and the results also supported that S. sinensis closely
clustered with S. oligofermentans and S. cristatus, forming a
unique clade (fig. 3A). Although this clade, comprising
S. sinensis, S. oligofermentans, and S. cristatus, also clustered
with members of sanguinis group, inclusion of this clade into
Genome Biol. Evol. 6(10):2930–2943. doi:10.1093/gbe/evu232 Advance Access publication October 20, 2014
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FIG. 3.—Phylogenetic tree constructed using draft genome sequences and concatenated nucleotide sequences of 50 ribosomal protein genes of S.
sinensis HKU4T. In (A), the tree based on draft genome sequences was constructed by neighbor-joining method using GGDC distance (formula 1) and E.
faecalis V583 as the root. In (B), the tree based on 50 ribosomal protein genes, showing the relationship of S. sinensis HKU4T to other Streptococcus species,
was constructed by maximum-likelihood method using RAxML (version 7.3) and E. faecalis V583 as the root. Bootstrap values were calculated from 1,000
replicates. The scale bar corresponds to the mean number of nucleotide substitutions per site on the respective branch. The gene names and accession
numbers are given as cited in GenBank in table 1.
the sanguinis group does not appear justified as they were
connected by a relatively long branch and shared relatively low
DDH value with S. sinensis. To confirm this result, we performed phylogenetic analysis using concatenated sequences
of 50 ribosomal protein genes retrieved from 87
Streptococcus genomes including S. sinensis, representing
35 different species, and from one E. faecalis genome.
These 50 ribosomal protein genes were chosen because
they have been shown to be useful for phylogenetic delineation of bacterial species in previous studies (Jolley et al. 2012;
Maiden et al. 2013) and their names were listed in supplementary table S2, Supplementary Material online. Results
showed that the topologies of both trees were concordant
and able to recover members of the seven taxonomic groups
as described in previous studies (fig. 3A and B) (Kawamura
et al. 1995; Facklam 2002). Consistently, the tree
based on the concatenated sequences also revealed that
S. sinensis, together with S. oligofermentans and S. cristatus,
forming a distinct phylogenetic clade with a bootstrap value
of 100%. It is worth noting that this unique clade also
fell within the sanguinis group but the grouping was weakly
supported by a low bootstrap value of 42%, meaning that
it was not often clustered with members of sanguinis group
(fig. 3B).
The phylogenetic position of S. sinensis has been considered controversial due to its simultaneous possession of
2938 Genome Biol. Evol. 6(10):2930–2943. doi:10.1093/gbe/evu232 Advance Access publication October 20, 2014
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Streptococcus sinensis HKU4T Genome Sequence
FIG. 3.—Continued.
phenotypic characteristics from members of mitis, sanguinis,
and anginosus groups (Woo et al. 2006). Genotypic methods
such as 16S rRNA or GroEL sequence analyses also failed to
reveal its exact taxonomic group. In this study, the genome
data, offering superior discriminatory power than any phenotypic and genotypic methods, resolve the taxonomic ambiguity of S. sinensis. Based on these genomic evidences, we
suggest the three species, S. sinensis, S. oligofermentans,
and S. cristatus, forming a well-supported clade, might best
be considered as a separate group that we tentatively propose
it as the sinensis group.
MALDI-TOF MS Analysis Supports the
Newly Proposed Sinensis Group
Dendrogram generated from hierarchical cluster analysis of
MALDI-TOF MS showed that the MALDI-TOF MS spectrum
of S. sinensis HKU4T and 28 nonduplicated Streptococcus species correctly recovered members of salivarius, mutans, bovis
and pyogenic groups, and Streptococcus suis (fig. 4).
Streptococcus sinensis consistently formed a distinct cluster
with S. oligofermentans and S. cristatus, but these three streptococci were clustered with members belonging to “mitis
Genome Biol. Evol. 6(10):2930–2943. doi:10.1093/gbe/evu232 Advance Access publication October 20, 2014
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FIG. 4.—Dendrogram generated from hierarchical cluster analysis of MALDI-TOF MS spectra of S. sinensis HKU4T and 28 isolates of other Streptococcus
species. Distances are displayed in relative units.
group” (fig. 4). Notably, in contrast to the previous
phylogenomic analysis using 50 concatenated genes or
whole genomes, these three streptococci were clustered
with “sanguinis group” (fig. 3A and B). On the basis of all
these findings, these three streptococci should not be included
into either sanguinis group or mitis group, but they should
exist as a novel group, sinensis group in the genus
Streptococcus.
Comparative Genomic Analyses
of the Three Genomes of Sinensis
Group
Based on BLAST (Basic Local Alignment Search Tool) analysis
using 50% nucleotide identity as a threshold, we compared
the genome sequence of S. sinensis HKU4T with those of S.
oligofermentans and S. cristatus and found that 1,241 CDSs
were shared among the three genomes (fig. 5). There were
222 CDSs only shared with S. oligofermentans and 63 CDSs
only shared with S. cristatus (fig. 5). A total of 466 CDSs were
uniquely found in S. sinensis HKU4T genome (fig. 5). Among
these 466 unique CDSs, only 90 CDSs could be classified
into RAST subsystems according to their predicted functional
roles. For the remaining 376 CDSs, which did not belong
to any subsystem, 256 CDSs were only annotated as hypothetical proteins. More in-depth analyses on these unique
CDSs, especially those annotated as hypothetical proteins,
may provide insights about the differences in metabolic
capacity between S. sinensis and two other members of
sinensis group.
Potential Virulence Factors in
S. sinensis HKU4T
Previous studies showed that S. sinensis has been recovered
from patients with infective endocarditis globally (Woo et al.
2002, 2004; Uckay et al. 2007; Faibis et al. 2008), suggesting
that the bacteria may possess virulence factors to adhere and
to colonize heart valves and to cause endocardial damage. The
draft genome of S. sinensis HKU4T contains homologs of several virulence genes that are important for the pathogenesis of
infective endocarditis. These include platelet activating factor,
clumping factor B, collagen-binding protein, laminin-binding
protein, and fibronectin/fibrinogen-binding protein which
have been implicated in the pathogenesis of infective endocarditis by promoting bacterial adherence to endothelial tissues and triggering inflammatory responses (supplementary
table S3, Supplementary Material online) (Abranches et al.
2011; Que and Moreillon 2011). Notably, two other members
of the sinensis group, S. oligofermentans and S. cristatus, have
also been isolated from patients with infective endocarditis
(Matthys et al. 2006; Matta et al. 2009). Detailed comparative
genomic studies on the S. sinensis genome and genomes of
members of the sinensis group and those of the mitis group,
including Streptococcus mitis, S. tigurinus, and Streptococcus
oralis, may shed light on the ecology and biology of S. sinensis
2940 Genome Biol. Evol. 6(10):2930–2943. doi:10.1093/gbe/evu232 Advance Access publication October 20, 2014
GBE
Streptococcus sinensis HKU4T Genome Sequence
FIG. 5.—Comparative genomic analysis among three members of sinensis group, including S. sinensis, S. oligofermentans, and S. cristatus. The total
number of CDSs per genome is given as indicated. The overlapping sections indicate shared numbers of CDSs among genomes.
as well as pathogenesis of infective endocarditis caused by
members belonging to this new phylogenetic clade.
Collection of Institut Pasteur or Culture Collection, University
of Göteborg (CCUG) (Woo et al. 2002).
Conclusions
Genome Sequencing, Assembly, and Annotation of
S. sinensis Type Strain HKU4T
In this study, we sequenced the first draft genome
of S. sinensis type strain HKU4T and unambiguously
determined the phylogenetic position of S. sinensis using phylogenomic approach. Phylogenomic and MALDI-TOF MS analysis revealed a distinct phylogenetic clade in the genus
Streptococcus, which we proposed it as sinensis group,
currently comprising three species, S. sinensis, S. oligofermentans, and S. cristatus. The present draft genome sequence has
also allowed rapid exploration of potential virulence genes in
S. sinensis. Our findings also illustrate the power of phylogenomic analyses for resolving ambiguities in bacterial
taxonomy.
Materials and Methods
Bacterial Strains
A total of 29 nonduplicated Streptococcus strains, including S.
sinensis HKU4T, were included in the MALDI-TOF MS analysis
(supplementary table S4, Supplementary Material online).
Streptococcus sinensis HKU4T was isolated from blood culture
of a Chinese patient with infective endocarditis in Hong Kong,
whereas the remaining strains were either purchased from the
The draft genome sequence of S. sinensis HKU4T was determined by high-throughput sequencing with Illumina Hi-Seq
2500. Genomic DNA was extracted from overnight cultures
(37 C) grown on blood agars by genomic DNA purification
kit (QIAgen, Hilden, Germany) as described previously
(Woo et al. 2009; Tse et al. 2010). It was sequenced
by 151-bp paired-end reads with mean library size of
350 bp. Sequencing errors were corrected by k-mer
frequency spectrum analysis using SOAPec (http://soap.genomics.org.cn/about.html, last accessed October 22, 2014). De
novo assembly was performed using SOAPdenovo2 (http://
soap.genomics.org.cn/soapdenovo.html,
last
accessed
October 22, 2014). Prediction of protein-coding regions and
automatic functional annotation was performed using
Glimmer3 and RAST server version 4.0 (Delcher et al. 2007;
Aziz et al. 2008). Intergenomic distance between S. sinensis
HKU4T and other Streptococcus species, including representatives from seven major groups, was calculated using GGDC
2.0 (http://ggdc.dsmz.de/distcalc2.php, last accessed October
22, 2014) (Auch et al. 2010). Streptococcus species included
for the distance calculation was shown in table 1.
Genome Biol. Evol. 6(10):2930–2943. doi:10.1093/gbe/evu232 Advance Access publication October 20, 2014
2941
GBE
Teng et al.
Genome Sequence Data
Details of the 88 genome sequences used in this study are
shown in table 1. Despite the 18 draft genome sequences,
including one from S. sinensis HKU4T which was sequenced to
near completion as part of this study, the remaining 70
genome sequences were complete and downloaded from
National Center for Biotechnology Information. Nucleotide sequences of 50 ribosomal protein genes (supplementary table
S2, Supplementary Material online) and one copy of 16S rRNA
and groEL genes were retrieved, respectively, from all 88 genomes (table 1).
Phylogenetic Characterization
The tree based on entire genome sequences was constructed
by neighbor-joining method using GGDC distance (formula 1:
length of all high-scoring segment pairs divided by total
genome length) and E. faecalis as the root. The trees based
on single gene loci, 16S rRNA and groEL gene (nucleotide and
amino acid), were aligned by Geneious 7.1.5 (Biomatters
Limited) and constructed, respectively, by maximum-likelihood
method using RAxML (version 7.3). The tree based on the
concatenated sequences of 50 ribosomal protein genes
from 88 Streptococcus species, including 35 nonduplicated
species, was constructed by maximum-likelihood method
using RAxML (version 7.3). A total of 20,921 nucleotide positions were included in the analysis. Nucleotide sequences of
corresponding homologs in E. faecalis were used as the
outgroups where appropriate.
Matrix-Assisted Laser Desorption Ionization Time-of-Flight
Mass Spectrometry
MALDI-TOF MS was performed as previously described with
slight modifications (Tang et al. 2013; Lau et al. 2014).
Twenty-nine nonduplicated Streptococcus strains, including
S. sinensis HKU4T, were grown on sheep blood agar at
37 C with 5% CO2 for 48 h. All isolates were analyzed by
the ethanol formic acid extraction method using the same
experimental conditions suggested by the Bruker system.
Briefly, 1–3 colonies were suspended into 100 ml of HPLC
grade water (Fluka, St Louis, MO). Then, 300 ml of absolute
ethanol was added and incubated for 5 min at room temperature. Cell pellet was then air dried after centrifugation. The
pellet was resuspended in 30 ml each of formic acid (Fluka) and
acetonitrile (Sigma Aldrich, St Louis, MO). Each bacterial extract was spotted onto six spots of the MSP96 target plate
after centrifugation. Samples were processed in the Bruker
MicroFlex LT mass spectrometer (Bruker Daltonics, Bremen,
Germany) with 1 ml of a-cyano 4-hydroxycinnimic acid
matrix solution (Sigma Aldrich). Spectra were obtained with
an accelerating voltage of 20 kV in linear mode and analyzed
within m/z range 3,000–15,000 Da. Spectra were analyzed
with MALDI Biotyper 3.1 and Reference Library V.3.1.2.0
(Bruker Daltonics). A mass spectrum profile (MSP) based on
24 separate determinations was created. The representative
MSPs were then selected for hierarchical cluster analysis using
MALDI Biotyper 3.1 (Bruker Daltonics) with default parameters
(Ketterlinus et al. 2005), where distance values are relative and
are always normalized to a maximum value of 1,000.
Supplementary Material
Supplementary tables S1–S4 are available at Genome Biology
and Evolution online (http://www.gbe.oxfordjournals.org/).
Acknowledgments
This work was partly supported by the Strategic Research
Theme Fund and the Small Project Funding Scheme,
The University of Hong Kong. The authors thank members of the Centre for Genomic Sciences, The University of
Hong Kong, for their technical support in genome
sequencing.
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Associate editor: B. Venkatesh
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